CHARACTERIZATION OF THE FOUR GENES ENCODING CYTOPLASMIC RIBOSOMAL PROTEIN S15a IN ARABIDOPSIS THALIANA A Thesis Submitted to the College of Graduate Studies and Research in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Department of Biology University of Saskatchewan Saskatoon By Jacqueline Louise Hulm © Copyright Jacqueline Louise Hulm, March, 2008. All rights reserved. PERMISSION TO USE In presenting this thesis in partial fulfillment of the requirements for a Postgraduate degree from the University of Saskatchewan, I agree that the Libraries of this University may make it freely available for inspection. I further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis work or, in their absence, by the Head of the Department or the Dean of the College in which my thesis work was done. It is understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis. Requests for permission to copy or to make other use of material in this thesis in whole or in part should be addressed to: Head of the Department of Biology 112 Science Place, University of Saskatchewan Saskatoon, Saskatchewan S7N 5E2 i ABSTRACT Eukaryotic cytosolic ribosomes are composed of two distinct subunits consisting of four individual ribosomal RNAs and, in Arabidopsis thaliana, 81 ribosomal proteins. Functional subunit assembly is dependent on the equimolar production of each ribosomal component. Arabidopsis thaliana r-protein genes exist in multi-gene families ranging in size from two to seven transcriptionally active members. The cytosolic RPS15a gene family consists of four members (RPS15aA, -C, -D and -F) that, at the amino acid level, share 87-100% identity. Using semi-quantitative RT-PCR I have shown that RPS15aC is not expressed and that transcript abundance differs both spatially and temporally among the remaining RPS15a genes in non-treated Arabidopsis tissues and in seedlings following a variety of abiotic stresses. A comprehensive analysis of the RPS15a 5' regulatory regions (RRs) using a series of deletion constructs was used to determine the minimal region required for gene expression and identify putative cis-regulatory elements. Transcription start site mapping using 5' RACE indicated multiple sites of initiation for RPS15aA and -F and only a single site for RPS15aD while all three genes contain a leader intron upstream of the start codon. Analysis of reporter gene activity in transgenic Arabidopsis containing a series of 5' RR deletion::GUS fusions showed that, similar to previous RT-PCR results, there was a trend for mitotically active tissues to stain for GUS activity. Putative cis-elements including the TELO box, PCNA Site II motif and pollen specific elements were identified. However, there was not always a clear correlation between the presence of a putative element and RPS15a transcript abundance or GUS activity. Although variation in transcriptional activity of each RPS15a gene has been observed, subcellular localization of both RPS15aA and -D in the nucleolus has been confirmed in planta by confocal microscopy. The results of this thesis research suggest while all three active RPS15a genes are transcriptionally regulated, additional post-transcriptional and/or translational regulation may be responsible for final RPS15a levels while differential isoform incorporation into ribosomal subunits may be the final point of r-protein regulation. ii ACKNOWLEDGEMENTS I would like to begin by thanking my supervisor Dr. Peta Bonham-Smith not only for her guidance, but also for sharing her knowledge and enthusiasm. It has been a sincere pleasure being a member of your lab. I would also like to thank the members of my advisory committee, Dr. Pierre Fobert, Dr. William Roesler and Dr. Kenneth Wilson. Your constant support, insight and advice have been invaluable. I would be remiss if I didn’t thank the many members of the departmental staff, especially Dennis Dyck, and Marlynn Mierau for their technical assistance as well as Shirley Hleck, Leona Leason, Joan Virgl and Deidre Wasyliw for all the work they have done on my behalf. My years in graduate school would not have been the same had I not shared them with colleagues I am lucky to also count as friends: Rebecca Cross, Rory Degenhardt, Robin Kusch, Donna Lindsay, Kerri McIntosh, Jeffrey Pylatuik, Kerry Sproule, Remko Verspui, Heather Wakely and Lester Young. Thank you for your support during the highs and inevitable lows of which graduate school is composed. Lastly, I wish to thank my parents, Doug and Sandra Hulm who have been with me for every step down this long and challenging path. For their love, encouragement and patience I will always be grateful. iii TABLE OF CONTENTS PERMISSION TO USE ..........................................................................................................i ABSTRACT........................................................................................................................ii ACKNOWLEDGEMENTS....................................................................................................iii TABLE OF CONTENTS.......................................................................................................iv LIST OF TABLES..............................................................................................................vii LIST OF FIGURES............................................................................................................viii LIST OF ABBREVIATIONS .................................................................................................. x 1. LITERATURE REVIEW.................................................................................................... 1 1.1. Introduction ....................................................................................................... 1 1.2. Historical Overview........................................................................................... 2 1.3. Basic Ribosome Structure ................................................................................. 8 1.3.1. rRNA ...................................................................................................... 11 1.3.1.1. rRNA genes (rDNA)...................................................................... 11 1.3.2. Ribosomal proteins................................................................................. 14 1.3.2.1. R-protein structure......................................................................... 14 1.3.2.2. Intra-ribosomal functions .............................................................. 15 1.3.2.2.1. mRNA recognition ............................................................... 15 1.3.2.2.2. tRNA interactions................................................................. 16 1.3.2.2.3. Peptide exit tunnel, signal recognition, secretion and chaperones ..................................................................... 16 1.3.2.3. Extra-ribosomal functions ............................................................. 17 1.4. R-Protein Gene Expression and Regulation.................................................... 17 1.4.1. Prokaryotes............................................................................................. 17 1.4.2. Organization of r-protein genes in eukaryotes ....................................... 18 1.4.3. Regulation of eukaryotic r-protein genes ............................................... 19 1.4.3.1. Transcriptional regulation ............................................................. 19 1.4.3.1.1. Saccharomyces cerevisiae.................................................... 19 1.4.3.1.2. Mammals .............................................................................. 20 1.4.3.1.3. Plants .................................................................................... 23 1.4.3.2. Post-transcriptional regulation....................................................... 24 1.5. Ribosome Heterogeneity and the Ribosome Filter Hypothesis....................... 26 1.6. R-protein S15a................................................................................................. 29 1.7. Objectives........................................................................................................ 30 2. VARIATION IN TRANSCRIPT ABUNDANCE AMONG THE FOUR MEMBERS OF THE ARABIDOPSIS THALIANA CYTOSOLIC RIBOSOMAL PROTEIN S15a GENE FAMILY ........... 31 2.1. Introduction ..................................................................................................... 32 iv 2.2. Materials and methods..................................................................................... 34 2.2.1. Plant material and seedling cultivation................................................... 34 2.2.2. Treatments .............................................................................................. 34 2.2.2.1. Wild type (non-treated) ................................................................. 34 2.2.2.2. Phytohormones.............................................................................. 34 2.2.2.3. Temperature stress......................................................................... 35 2.2.2.4. Wounding ...................................................................................... 35 2.2.2.5. Copper sulfate stress.....................................................................